The proposed research project focuses on the molecular basis of regulation of the ATP-sensitive potassium channel (K/ATP). The K/ATP potassium inward rectifiers, composed of four pore forming subunits (Kir6.x) and four regulatory subunits (SURx) to form a hetero-octamer, have been identified in cardiac muscle, vascular smooth muscle, nonvascular smooth muscle, pancreatic beta cells, skeletal muscle, and neurons. Cardiac vascular smooth muscle K/ATP channel activity is enhanced through protein kinase A (PKA) activation. Direct phosphorylation of the K/ATP subunits has not been demonstrated and the regulatory role these potentially phosphorylated subunits play remains unresolved. The molecular basis of PKA regulation of K/ATP will be addressed by the following specific aims; 1) The physiological response of K/ATP to PKA stimulation will be examined at both the whole cell and single channel level using electrophysiological studies of co-expressed Kir6.x and SURx. 2) Direct phosphorylation of K/ATP will be investigated and the sites identified by in vitro phosphorylation of immunoprecipitated subunits. 3) The individual contributions of the K/ATP subunits to regulation by PKA will be determined by electrophysiological studies of phosphorylation deficient mutants. K/ATP channels play an important role in regulating the coronary vascular smooth muscle resting membrane potential. Examination of the role PKA phosphorylation of K/ATP plays in regulating channel activity will provide insight into the regulation of cardiac function and provide a basis for the development of improved treatments for cardiovascular disease.